1. Field of the Invention
The present invention relates to a method for differentiating human peripheral blood granulocytes, and, more particularly, to a method for determining human peripheral blood granulocytes whether in a healthy person, or in those suffering from serious infectious diseases or inflammatory diseases, and the like.
2. Description of the Background Art
There have been practically no accurate methods for diagnosing or reliable methods for monitoring the patients who are suffering from an acute, subacute, or chronic inflammatory disease or a serious infectious disease, such as chronic tonsillitis, Crohn""s disease, agammaglobulinemia, ulcerative colitis, septicemia, leukemia, leukocyte adherence deficiency; or those who had undergone a kidney transplant. Development of an accurate diagnostic method and a monitoring method which can easily be applied for the evaluation of the curative effects for those patients have therefore been in demand.
In view of this situation, the present inventors have undertaken extensive studies on the relationship between the features of human peripheral blood granulocytes and various inflammatory diseases, and have found that an IgG.Fc receptor I (hereinafter referred to as xe2x80x9cIgG.FcRIxe2x80x9d) is scarcely present on the surface of human peripheral blood granulocytes of healthy persons, whereas it is evidently recognized on those of patients suffering from inflammatory diseases or the like. Further investigations have revealed that the IgG.FcRI can be recognized in vitro by acting an interferon on peripheral blood granulocytes. Based on this fact, it has been elucidated that IgG.FcRI on human peripheral blood granulocytes can be quantified by a method which comprises i) cultivating the granulocytes in the presence of an interferon, ii) acting an immune complex on the granulocytes in the presence of a chemiluminescent substance, and to induce IgG.FcRI presence on the granulocytes, and iii) measuring the intensified photon quantity generated by the stimulation of IgG.FcRI appearing on the granulocytes using a chemiluminescence method (hereinafter referred to as xe2x80x9cCLxe2x80x9d). This differentiation method is believed to contribute to the diagnoses of patients suffering from either an serious infectious disease or any one of the inflammatory diseases, and is advantageous for monitoring the curative effects of those patients. These findings have led to the completion of the present invention.
Accordingly, an object of the present invention is to provide a method for differentiating human peripheral blood granulocytes which comprises: cultivating the granulocytes in the presence of an interferon, acting an immune complex on the granulocytes in the presence of a chemiluminescent substance, and measuring the chemiluminescent quantities induced.
In a preferred embodiment, a human interferon-gamma is used as said interferon, and a complex containing mouse IgG is used as said immune complex.
Other objects, features and advantages of the invention will hereinafter become more readily apparent from the following description.
The human peripheral blood granulocytes to be used in the present invention can be human peripheral blood as is, or granulocytes separated from peripheral blood. Separation of human peripheral blood granulocytes from human peripheral blood can be carried out according to a known method, for example, stratifying heparin blood or EDTA blood in a xe2x80x9cMono-poly resolving mediumxe2x80x9d and subjecting it to centrifugation.
xe2x80x9cMono-poly resolving medium xe2x80x9d is a trademark of Flow Lab. Co., and it names a medium which can resolve the mono- form from the poly- form.
As an interferon (hereinafter referred to as xe2x80x9cIFNxe2x80x9d) used in the present invention, a human IFN-gamma is preferable, which includes those prepared from human lymphocytes, as well as recombinant IFN obtained by gene recombination techniques.
Luminol, lucigenin, luciferin, or the like, and their derivatives can be given as examples, but not limiting thereof, of the chemiluminescent substances to be used in the present invention.
The immune complexes used in this invention include a complex containing mouse IgG, for example, a complex in which mouse IgG and sheep red blood cells (hereinafter referred to as xe2x80x9cSRBCxe2x80x9d) are bonded chemically, a mixture of anti-SRBC-mouse IgG and SRBC, a complex of mouse IgG and latex, a complex of mouse IgG and various proteins, a complex of mouse IgG and various saccharides or polysaccharides, and the like. Specifically, a monoclonal or polyclonal mouse IgG2a is particularly preferable as the mouse IgG to be used.
In the practice of this invention, the human peripheral blood or the granulocytes thereof is first dispersed in an RPMI medium which contains 15% FCS, and then 10-1000 U/ml, preferably 100 U/ml, of IFN is added to the dispersion, followed by cultivation for 10-20 hours. To this culture, an appropriate amount of a chemiluminescent substance, which is dissolved in dimethylsulfoxide or the like and being diluted with a phosphate saline buffer (hereinafter referred to as xe2x80x9cPBSxe2x80x9d), and an immune complex are added in order to measure the quantity of chemiluminescence induced. In this instance, luminol is added as a preferable chemiluminescent substance at a concentration between 1xc3x9710xe2x88x923xe2x88x921xc3x9710xe2x88x926M and an IgG-SRBC complex as a preferable immune complex in an amount equivalent to 30 times to the number of granulocytes.
Differentiation of the subject granulocytes can be achieved by comparing the chemiluminescent values of those IFN-added and IFN-not added. For instance, the ratio of chemiluminescence amounts (hereinafter referred to as xe2x80x9cCL ratioxe2x80x9d) can be calculated according to the following equation:       CL    ⁢          xe2x80x83        ⁢    ratio    =                                          Peak            ⁢                          xe2x80x83                        ⁢            luminescence            ⁢                          xe2x80x83                        ⁢            counts            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            the            ⁢                          xe2x80x83                        ⁢            culture                                                            in            ⁢                          xe2x80x83                        ⁢            which            ⁢                          xe2x80x83                        ⁢            IFN            ⁢                          xe2x80x83                        ⁢            was            ⁢                          xe2x80x83                        ⁢            not            ⁢                          xe2x80x83                        ⁢            added                                                                    Peak            ⁢                          xe2x80x83                        ⁢            luminescence            ⁢                          xe2x80x83                        ⁢            counts            ⁢                          xe2x80x83                        ⁢            of            ⁢                          xe2x80x83                        ⁢            the            ⁢                          xe2x80x83                        ⁢            culture                                                            in            ⁢                          xe2x80x83                        ⁢            which            ⁢                          xe2x80x83                        ⁢            IFN            ⁢                          xe2x80x83                        ⁢            was            ⁢                          xe2x80x83                        ⁢            added                              
In this equation, a large CL ratio indicates that the granulocytes inspected are those of patients suffering from inflammation or the like, whereas a small CL ratio denotes that the granulocytes inspected are those of healthy person.